Samples
Ejaculates containing at least 20 x 106 spermatozoa/ml and showing at least 50% progressive sperm motility were obtained from 18 healthy men by masturbation, after a minimum of 48 h of sexual abstinence. Informed consent was obtained from each donor. Semen analysis was performed according to the guidelines published by the World Health Organization (1999
). Each ejaculate was swim-up prepared (SUP) and divided into four aliquots for: conventional slow freezing with (CSF+) or without (CSF–) standard cryoprotectants, and vitrification with (V+) or without (V–) standard cryoprotectants. The cryoprotectants used were glycerol/egg yolk. Swim-up was performed using a standard medium containing 10 mg/ml of human serum albumin (SPM; Scandinavian IVF Science, Gothenburg, Sweden) according to the instructions published by the World Health Organization (1999). In short, an ejaculate was washed twice by centrifugation at 380 g for 10 min in a double volume of SPM. After the second washing, 0.8 ml of SPM were pipetted over the pellet. The samples were then incubated for 30 min for swim-up.
Fresh SUP spermatozoa (no cryoprotectant) served as controls for all the experimental groups.
Conventional (automated) freezing
For the conventional, programmable slow freezing method, the cryoprotectant used was test–egg yolk–glycerol (TEYG) freezing medium (Scandinavian IVF Science, Gothenburg, Sweden). After 1:1 dilution in TEYG (final glycerol concentration 6%), 0.25 ml of the spermatozoa suspension was pipetted into standard 0.25 ml insemination straws (MTG, Altdorf, Germany) and kept at room temperature for 10 min. The straws were then placed in a programmable freezer.
Semen samples in both groups (CSF+ and CSF–) were frozen according to Giraud et al. (2000). The protocol for conventional freezing was the following: cooling from 22 to 4°C at a rate of 5°C/min; from 4 to –30°C at a rate of 10°C/min; and from –30 to –140°C at a rate of 20°C/min, followed by plunging into liquid nitrogen. After storage of the spermatozoa in liquid nitrogen for a minimum of 24 h, the samples were thawed by plunging the straws into a water bath at 37°C for 50 s. Next, 5 ml of SPM were added to the thawed samples and the sperm suspension was centrifuged at 380 g for 5 min. The supernatant was removed and the pellet was resuspended in 100 µl of SPM.
Cryopreservation by direct plunging into liquid nitrogen (vitrification)
The method of vitrification used was described in detail by Nawroth et al. (2002). Briefly, the same concentration of TEYG as for slow freezing was used for vitrification in the presence of a cryoprotectant. Drops (20 ± 2 µl) of sperm samples in both groups (V+ and V–) were placed on copper loops of 5 mm diameter. These cryoloops were then plunged into liquid nitrogen and stored for at least 24 h. After the storage period, the samples were warmed by plunging the copper loops into a 15 ml tube containing 10 ml of SPM at 37°C and mixing thoroughly. After warming five loops per tube, the tubes were placed in a CO2 incubator for 5–10 min. The spermatozoa were then concentrated by centrifugation at 380 g for 10 min. The pellet was resuspended in 100 µl of SPM.
Evaluation of sperm motility and viability
Sperm motility was assessed immediately after liquefaction (conventional freezing) or sample concentration by centrifugation (vitrification). The Makler chamber was used for motility scoring. Motility was estimated under the light microscope using the x400 magnification. Only spermatozoa with progressive motility (WHO categories ‘a’ and ‘b’) were assessed. Motility was evaluated immediately after thawing. Recovery of motile spermatozoa was defined as the percentage of post-thaw motility x 100% divided by the percentage of pre-freezing motility. To test the effect of the cryoprotectant on sperm motility and DNA integrity before cryopreservation, spermatozoa suspensions were equilibrated in TEYG for 10 min and then washed with SPM for swim-up preparation.
Comet assay
The comet assay was performed using the CometAssayTM Reagent Kit for Single Cell Gel Electrophoresis Assay (Trevigen, Inc., Gaithersburg, MD) according to the manufacturer’s instructions with slight modification by Donnelly et al. (2001b). Briefly, the spermatozoa samples were washed twice with SPM and the sediment was resuspended in Dulbecco’s phosphate-buffered saline (Ca2+- and Mg2+-free PBS; Bio-Wittaker, Verviers, Belgium). The samples were then placed on ice to inhibit endogenous damage occurring during sample preparation. During preparation, the cells were handled under yellow light to prevent DNA damage by UV light. Some cells were treated with 25 µmol/l KMnO4 for 20 min at 4°C, as controls for the comet assay (sperm cells with a comet tail have disrupted DNA). Subsequent treatment of DNA-damaged and undamaged cells was performed as follows. Freshly prepared lysis solution supplemented with 1% dimethylsulphoxide (DMSO) was chilled at 4°C for at least 20 min before use. The lysis solution contained 2.5 mol/l sodium chloride, 100 mmol/l EDTA pH 10, 10 mmol/l Tris base, 1% sodium lauryl sarcosinate and 1% Triton X-100. After mixing the spermatozoa suspension (at 
1 x 105 cells/ml) with 1% molten low-melting point agarose at 40°C at a ratio of 1:10 (v/v), 75 µl of suspension was immediately pipetted onto the Trevigen CometSlideTM, gently spread over the slide area and placed flat in the dark at 4°C for 10 min. The slides were then immersed in the pre-chilled lysis solution for 60 min for dissolution of the cell membranes. To achieve DNA decondensation after cell lysis, the slides were incubated with 10 mmol/l dithiothreitol (DTT; Sigma-Aldrich, Steinheim, Germany) for 30 min at 4°C and then with 4 mmol/l 3.5-diodosalicylic acid lithium salt (LIS, Sigma-Aldrich) for 90 min at 20°C. After tapping the slides to remove excess solution, they were immersed in freshly prepared alkaline solution (300 mmol/l NaOH, 1 mmol/l EDTA, pH >13) in the dark for 20 min at room temperature. A horizontal gel electrophoresis apparatus was filled with the same alkaline solution at 4°C. The slides were placed flat onto a gel tray and aligned equidistant from the electrodes. Electrophoresis was performed at 1 V/cm adjusted to 300 mA by either raising or lowering the buffer level in the apparatus for 10 min. After electrophoresis, the excess solution was gently tapped from the slides, which were then dipped in 70% ethanol for 5 min with subsequent air-drying at room temperature before being stored in an airtight desiccator. The slides were viewed using a Zeiss IM epifluorescence microscope equipped with an excitation/emission filter of 485 nm/520 nm under x400 magnification. Fluorescent staining was performed using SYBR green stain (working concentration 1:200). In healthy cells, the fluorescence was confined to the nucleoid: undamaged DNA is supercoiled and does not migrate very far from the nucleoid (Figure 1). In cells that have incurred damage to the DNA, the alkali treatment unwinds the DNA, releasing fragments that migrate from the nucleoid (Figure 2). A total of 200 cells were analysed per slide.
Evaluation of sperm morphology
Sperm morphology was assessed using strict criteria (Menkveld et al., 1991). The sperm were stained using Testsimplets (Roche Diagnostics LTD, Germany). After pre-staining slides with methylene blue and cresyl violet acetate, 5 µl of sperm were dropped onto the centre of a pre-stained slide and covered with a coverglass. Morphological assessment was performed using an oil immersion microscope at x1000 magnification after 30 min of staining. Results were recorded as the number of normal spermatozoa out of 100 counted on each slide.
Statistical analysis
Treatment effects on sperm variables were assessed by ANOVA. Data are expressed as means ± SD. The level of statistical significance was set at P 
0.05.
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